Akira Tomie

Genome-wide DNA methylation analysis in hepatocellular carcinoma

Epigenetic changes as well as genetic changes are mechanisms of tumorigenesis. We aimed to identify novel genes that are silenced by DNA hypermethylation in hepatocellular carcinoma (HCC). We screened for genes with promoter DNA hypermethylation using a genome-wide methylation microarray analysis in primary HCC (the discovery set). The microarray analysis revealed that there were 2,670 CpG sites that significantly differed in regards to the methylation level between the tumor and non-tumor liver tissues; 875 were significantly hypermethylated and 1,795 were significantly hypomethylated in the HCC tumors compared to the non‑tumor tissues. Further analyses using methylation-specific PCR, combined with expression analysis, in the validation set of primary HCC showed that, in addition to three known tumor-suppressor genes (APC, CDKN2A, and GSTP1), eight genes (AKR1B1, GRASP, MAP9, NXPE3, RSPH9, SPINT2, STEAP4, and ZNF154) were significantly hypermethylated and downregulated in the HCC tumors compared to the non-tumor liver tissues. Our results suggest that epigenetic silencing of these genes may be associated with HCC.

Linked color imaging improves endoscopic diagnosis of active Helicobacter pylori infection.

Background and study aims: Linked color imaging (LCI) is a new image-enhanced endoscopy technique using a laser light source to enhance slight differences in mucosal color. The aim of this study was to compare the usefulness of LCI and conventional white light imaging (WLI) endoscopy for diagnosing Helicobacter pylori (H. pylori). Patients and methods: We retrospectively analyzed images from 60 patients examined with WLI and LCI endoscopy between October 2013 and May 2014. Thirty patients had H. pylori infections, and other thirty patients tested negative for H. pylori after eradication therapy. Four endoscopists evaluated the 2 types of images to determine which was better at facilitating a diagnosis of H. pylori infection. Results: H. pylori infection was identified with LCI by enhancing the red appearance of the fundic gland mucosa. The accuracy, sensitivity, and specificity for diagnosing H. pylori infection using WLI were 74.2 %, 81.7 %, and 66.7 %, respectively, while those for LCI were 85.8 %, 93.3 %, and 78.3 %, respectively. Thus, the accuracy and sensitivity for LCI were significantly higher than those for WLI (P = 0.002 and P = 0.011, respectively). The kappa values for the inter- and intraobserver variability among the 4 endoscopists were higher for LCI than for WLI. Conclusions: H. pylori infection can be identified by enhancing endoscopic images of the diffuse redness of the fundic gland using LCI. LCI is a novel image-enhanced endoscopy and is more useful for diagnosing H. pylori infection than is WLI.

Blue Laser Imaging-Bright Improves Endoscopic Recognition of Superficial Esophageal Squamous Cell Carcinoma

Background/Aims. The aim of this study was to evaluate the endoscopic recognition of esophageal squamous cell carcinoma (ESCC) using four different methods (Olympus white light imaging (O-WLI), Fujifilm white light imaging (F-WLI), narrow band imaging (NBI), and blue laser imaging- (BLI-) bright). Methods. We retrospectively analyzed 25 superficial ESCCs that had been examined using the four different methods. Subjective evaluation was provided by three endoscopists as a ranking score (RS) of each image based on the ease of detection of the cancerous area. For the objective evaluation we calculated the color difference scores (CDS) between the cancerous and noncancerous areas with each of the four methods. Results. There was no difference between the mean RS of O-WLI and F-WLI. The mean RS of NBI was significantly higher than that of O-WLI and that of BLI-bright was significantly higher than that of F-WLI. Moreover, the mean RS of BLI-bright was significantly higher than that of NBI. Furthermore, in the objective evaluation, the mean CDS of BLI-bright was significantly higher than that of O-WLI, F-WLI, and NBI. Conclusion. The recognition of superficial ESCC using BLI-bright was more efficacious than the other methods tested both subjectively and objectively.

EVI1, a target gene for amplification at 3q26, antagonizes transforming growth factor‐β‐mediated growth inhibition in hepatocellular carcinoma

EVI1 (ecotropic viral integration site 1) is one of the most aggressive oncogenes associated with myeloid leukemia. We investigated DNA copy number aberrations in human hepatocellular carcinoma (HCC) cell lines using a high‐density oligonucleotide microarray. We found that a novel amplification at the chromosomal region 3q26 occurs in the HCC cell line JHH‐1, and that MECOM (MDS1 and EVI1 complex locus), which lies within the 3q26 region, was amplified. Quantitative PCR analysis of the three transcripts transcribed from MECOM indicated that only EVI1, but not the fusion transcript MDS1–EVI1 or MDS1, was overexpressed in JHH‐1 cells and was significantly upregulated in 22 (61%) of 36 primary HCC tumors when compared with their non‐tumorous counterparts. A copy number gain of EVI1 was observed in 24 (36%) of 66 primary HCC tumors. High EVI1 expression was significantly associated with larger tumor size and higher level of des‐γ‐carboxy prothrombin, a tumor marker for HCC. Knockdown of EVI1 resulted in increased induction of the cyclin‐dependent kinase inhibitor p15INK4B by transforming growth factor (TGF)‐β and decreased expression of c‐Myc, cyclin D1, and phosphorylated Rb in TGF‐β‐treated cells. Consequently, knockdown of EVI1 led to reduced DNA synthesis and cell viability. Collectively, our results suggest that EVI1 is a probable target gene that acts as a driving force for the amplification at 3q26 in HCC and that the oncoprotein EVI1 antagonizes TGF‐β‐mediated growth inhibition of HCC cells.

Aberrant expression of the PHF14 gene in biliary tract cancer cells.

DNA copy number aberrations in human biliary tract cancer (BTC) cell lines were investigated using a high-density oligonucleotide microarray. A novel homozygous deletion was detected at chromosomal region 7p21.3 in the OZ cell line. Further validation experiments using genomic PCR revealed a homozygous deletion of a single gene, plant homeodomain (PHD) finger protein 14 (PHF14). No PHF14 mRNA or protein expression was detected, thus demonstrating the absence of PHF14 expression in the OZ cell line. Although the PHD finger protein is considered to be involved in chromatin-mediated transcriptional regulation, little is known about the function of PHF14 in cancer. The present study observed that the knock down of PHF14 using small interfering RNA (siRNA) enhanced the growth of the BTC cells. These observations suggest that aberrant PHF14 expression may have a role in the tumorigenesis of BTC.

Loss of PAR-3 protein expression is associated with invasion, lymph node metastasis, and poor survival in esophageal squamous cell carcinoma

Disrupted cell polarity is a feature of epithelial cancers. The partitioning defective 3 (PAR-3) protein, a key component of the PAR complex that regulates the polarization of cells, is involved in tight junction formation at epithelial cell-cell contacts. Our previous study detected a homozygous deletion of the PAR-3 gene in esophageal squamous cell carcinoma (ESCC) cell lines and frequent copy number loss of the PAR-3 gene in primary ESCC. Here, we aimed to investigate the clinicopathological relevance of altered expression of the PAR-3 protein in primary ESCC. We immunohistochemically analyzed expression of the PAR-3 protein, as well as that of other tight junction proteins, ZO-1 and claudin-1, in 74 primary ESCCs. While the PAR-3 protein was expressed in the cytoplasm of basal cells, it was localized on the plasma membrane of suprabasal cells of normal squamous epithelium of the esophagus. Of the 74 ESCC tumors, 20 (27%), 11 (15%), and 13 (18%) were negative for PAR-3, ZO-1, and claudin-1 proteins, respectively. Negative PAR-3 protein expression, but not negative ZO-1 or claudin-1 expression, was significantly associated with deeper tumor invasion (P<.01), positive lymph node metastasis (P=.03), and advanced tumor stage (P=.01). Patients with PAR-3-negative tumors showed marginally significantly shorter overall survival after surgery than those with PAR-3-positive tumors (P=.053). In conclusion, these results suggest that PAR-3 protein expression is frequently lost in primary ESCC and that loss of the PAR-3 protein is associated with aggressive clinicopathological features of ESCC.

Magnifying Endoscopy with Blue Laser Imaging Improves the Microstructure Visualization in Early Gastric Cancer: Comparison of Magnifying Endoscopy with Narrow-Band Imaging

Backgrounds Magnifying endoscopy with blue laser imaging (ME-BLI) for diagnosis of early gastric cancer (EGC) is as effective as magnifying endoscopy with narrow-band imaging (ME-NBI). However, there are different EGCs in microstructure visualization between ME-BLI and ME-NBI. This study aimed to clarify the pathological features of the EGCs, in which microstructure visualization was different between ME-NBI and ME-BLI. Methods EGCs were classified into groups A (irregular microsurface pattern (MSP) in ME-BLI and absent MSP in ME-NBI), B (irregular MSP in two modalities), or C (absent MSP in two modalities), according to the vessel plus surface classification. We compared the pathological features of EGCs between the three groups. Results 17, four, and five lesions could be evaluated in detail in groups A, B and C, respectively. Well-differentiated adenocarcinomas with shallow crypts were more frequent in group A than in group B (58.8 and 0%, resp.). The mean crypt depth of group A was significantly shallower than that of group B (56 ± 20, 265 ± 64 μm, resp., P = 0.0002). Conclusions ME-BLI could better visualize the microstructures of the EGCs with shallow crypts compared with ME-NBI. Therefore, ME-BLI could enable a more accurate diagnosis of EGC with shallow crypts.

Clinical and Pathological Challenges in the Diagnosis of Gastric-Type Differentiated Adenocarcinoma in the Stomach: A Study of Endoscopic Submucosal Dissection Cases

Background/Aims: Gastric-type differentiated adenocarcinoma (GDA) of the stomach is a rare variant of gastric cancer that is highly infiltrating and exhibits early metastasis. However, the endoscopic and pathological features of “early-stage” GDA remain unknown. The aim of this study is to characterize early-stage GDA. Methods: We retrospectively enrolled 479 differentiated-type early gastric cancer cases who underwent endoscopic submucosal dissection (ESD). GDA cases were selected based on morphology and immunohistochemistry. Clinicopathological data were compared between gastric- and intestinal-type differentiated adenocarcinomas (IDAs). Results: Thirteen lesions were classified as GDAs. GDAs as well as IDAs showed irregular microvascular and microsurface patterns with clear demarcation line on magnifying endoscopy with narrow band imaging (M-NBI). The rate of pathological misdiagnosis of GDAs in biopsy specimens was higher than that of IDAs (p = 0.016). GDA was significantly associated with positive lymphovascular invasion (p = 0.016). There was one intramucosal lesion with lymphatic invasion in GDA. Conclusions: Although M-NBI is useful to detect GDA, the pathological diagnosis of GDAs in biopsy specimens often remains challenging. When suspicious lesions are not diagnosed as GDA, they should be followed up intensively, or diagnostic ESD has to be performed. ESD specimens should be carefully evaluated because of a higher incidence of lymphovascular invasion.

Abstract 2539: MiR-96-5p functions as an oncogenic miRNA by inhibiting apoptosis in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the most aggressive cancers with high mortality worldwide. MicroRNAs (miRNAs) are small non-coding RNAs that have been used as cancer-related biomarkers and expected to be therapeutic agents. We performed genome-wide miRNA expression profiling of paired HCC tumors and non-tumorous liver tissues from patients with primary HCCs using the miRNA microarray (Agilent). We found that miR-96-5p was most significantly up-regulated in HCC tumors compared to non-tumor tissues. Although miR-96-5p is suggested to be an oncogenic miRNA, the function of miR-96-5p remains largely unknown. We identified the caspase-9 gene (CASP9) as a novel target of miR-96-5p, in addition to the forkhead box O1 gene (FOXO1) which is the known target of it. Caspase-9 protein is thought to play a central role in apoptosis and to be a tumor suppressor. Overexpression of miR-96-5p decreased caspase-9 protein expression and resulted in resistance to apoptosis induced by doxorubicin and UV in HCC cells. Our results suggested that miR-96-5p functions as an oncogenic miRNA by inhibiting apoptosis through decreasing caspase-9 expression in HCC. Citation Format: Naoto Iwai, Kohichiroh Yasui, Akira Tomie, Kei Teasaki, Tomoko Kitaichi, Osamu Dohi, Yasuyuki Gen, Yoshito Ito. MiR-96-5p functions as an oncogenic miRNA by inhibiting apoptosis in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2539. doi:10.1158/1538-7445.AM2017-2539

Abstract 1099: Up-regulated miR-96-5p inhibits apoptosis by targeting FOXO1 in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the most common cancers and the third leading cause of cancer death worldwide, especially in Asia and sub-Saharan Africa. MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression and can act as tumor suppressors or oncogenes. To identify miRNA involved in HCC, we performed a genome-wide miRNA gene expression analysis using the human miRNA microarray (Agilent) in paired tumor and non-tumor tissues from patients with primary HCC. The array-based miRNA expression profiles were validated by quantitative PCR. We identified miR-96-5p as the most significantly upregulated miRNA in HCC tumors. Although miR-96-5p has been recognized as an oncogenic miRNA, its role in the pathogenesis of HCC remains unknown. Our study showed that the inhibition of miR-96-5p reduced proliferation of HCC cells and induced apoptosis. Furthermore, we found that the forkhead box O1 (FOXO1) gene was a target of miR-96-5p. FOXO1, a transcription factor, plays an essential role in cell fate decisions. FOXO1 can induce apoptosis through mitochondria-dependent and mitochondria-independent pathways. Collectively, our results suggested that miR-96-5p may function as an oncogenic miRNA through inhibiting apoptosis by targeting FOXO1 in HCC. Citation Format: Naoto Iwai, Kohichiroh Yasui, Akira Tomie, Tomoko Kitaichi, Osamu Dohi, Yasuyuki Gen, Yoshito Itoh. Up-regulated miR-96-5p inhibits apoptosis by targeting FOXO1 in hepatocellular carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1099.